Hematopoietic stem cells (HSCs) are stem cells that form blood and immune cells and are ultimately responsible for the constant renewal of blood in an organism throughout the lifetime of the organism. HSCs have been the focus of research for many decades and are now routinely used for many therapeutic applications, including treatment for leukemia, lymphoma, inherited blood disorders, and HSC rescue after extensive chemotherapy regimens, among others.
HSCs are the best characterized example of a multipotential stem cell existing in tissues of an adult organism. In seminal studies by Trentin and colleagues (Trentin, 1965, Cardiovasc. Res. Cent. Bull 4:38-4; Till & McCulloch, 1961, Rad. Res. 14:213-222) lethally-irradiated mice died because they failed to replenish their circulating blood cells. However, transplantation of bone marrow cells from syngeneic donor animals rescued the host animal. The donor cells were responsible for repopulating all of the circulating blood cells. More recently, it has been shown that the cells responsible for reconstituting hematopoiesis in humans receiving a bone marrow transplant reside in a subset of cells expressing the CD34 antigen (CD34+) (Berenson et al., 1991, Blood 77:1717-1722). In other words, CD34+ cells comprise hematopoietic stem cells. Other methods of identifying and isolating populations enriched for hematopoietic stem cells that do not rely on cell surface immunophenotypes have been pursued. Primitive human hematopoietic progenitors have been isolated based on aldehyde dehydrogenase (ALDH) activity (Storms et al., 1999, Proc. Natl. Acad. Sci. 96:9118-9123; Hess et al., 2004, Blood 104:1648-1655)). In addition, engraftment in humans is highly correlated with the number of cells infused having ALDH activity (ALDHbr) and low side scatter (SSClo) (Fallon et al., 2003, Br. J. Haematol. 122:99-108).
A wealth of elegant studies have demonstrated that donation of a finite number of undifferentiated hematopoietic stem cells is capable of regenerating each of the eight or more different blood cell lineages in a host animal. This large body of work has provided the basis for bone marrow transplantation, a widely accepted therapeutic modality for cancer and inborn errors of metabolism. Thus, hematopoietic stem cells remain present in normal human bone marrow throughout life; they are not limited to the neonatal period.
There are currently three major sources of hematopoietic stem cells (HSCs) used for the purpose of bone marrow transplantation: adult bone marrow, adult peripheral blood, and the mononuclear fraction of umbilical cord blood following child birth. Cells isolated for the purpose of transplantation are typically isolated from the bone marrow or peripheral blood of adults. Disadvantageously, however, CD34+ cells are extremely rare in adult bone marrow (˜1-2%), requiring a significant volume of marrow to be obtained to generate a sufficient quantity of HSCs to engraft an adult. The procedure to obtain bone marrow is a lengthy and unpleasant procedure for the marrow donor, requiring extended hospitalizations to allow for bone marrow recovery. Isolation of CD34+ or ALDHbr cells from peripheral blood, typically requiring multiple cytapheresis sessions, is less unpleasant for the donor, however, it requires pre-inoculation of the donor with cytokines to mobilize the HSCs to the peripheral blood. Despite the mobilization, the number of CD34+ or ALDHbr cells, and consequently, HSCs, in peripheral blood is still typically quite low.
Umbilical cord blood (UCB) is somewhat more enriched for CD34+ cells, compared to bone marrow or mobilized peripheral blood (Wang et al., 1997, Blood 89:3919-3924), but the small volume of blood available in an umbilical cord limits the absolute number of engraftable HSCs that can be recovered, making UCB less useful than originally anticipated. In theory, one could provide a sufficient number of HSCs by pooling multiple umbilical cord blood samples, however, pooling is frowned on in clinical practice. Thus, cord blood-derived CD34+ cells are limited in their utility in adults because the consequential low numbers of HSCs isolated are too few to successfully engraft a full-grown adult. Expansion protocols have been studied to try and increase the cell numbers. Typically, however HSC expansion is accompanied by substantial differentiation, which is not always desirable.
There is a growing body of evidence that hematopoietic progenitors may not be limited to the bone marrow microenvironment. Investigators at University of Calgary have examined neuronal stem cells, which routinely differentiate along neuronal cell lineage pathways. When these cells were transplanted into lethally-irradiated hosts, the investigators detected the presence of donor cell markers in newly-produced myeloid and lymphoid cells (Bjornson, 1999, Science 283:534-537). Investigators at Baylor College of Medicine have performed similar studies using satellite cells isolated from murine skeletal muscle (Jackson et al., 1999, PNAS 96:14482-14486). When these muscle-derived cells were transplanted into lethally-irradiated hosts, the investigators detected the presence of the muscle gene markers in all blood cell lineages. Together, these studies indicate that neuronal and muscle tissues contain stem cells capable of hematopoietic differentiation. This suggests that sites other than the bone marrow may provide a renewable source of hematopoietic progenitors with potential application to human disease therapy (Quesenberry et al., 1999, J. Neurotrauma 16:661-666: Scheffler et al., 1999, Trends Neurosci 22:348-357; Svendsen & Smith, 1999, Trends Neurosci 22:357-364).
More recently, adipose-derived stromal vascular fraction (SVF) cells have been shown to successfully reconstitute major hematopoietic lineages in lethally-irradiated mice (Cousin et al., 2003, Biochem. Biophys. Res. Commun. 301:1016-1022 and U.S. Patent Publication No. 2004/0067218). Mesenchymal stromal stem cells, isolated from adipose tissue, have also been co-infused with CD34+ cells, isolated from mobilized peripheral blood, and were found to facilitate successful engraftment of the CD34+ cells (Kim et al., 2005, Biochem. Biophys. Res. Commun. 329(1):25-31).
U.S. Patent Publication No. 2001/0033834 discloses an isolated adipose-derived stromal cell that has been differentiated to express at least one characteristic of a hematopoietic progenitor cell. Also disclosed are adipose-derived stromal cells that are de-differentiated into fully functional pleuripotent stem cells that can then be differentiated into hematopoietic cell lineages.
Despite the work summarized above, there remains a need for an abundant source of hematopoietic stem cells for use in therapeutic and other applications. The present invention addresses and meets these needs.